Preparation and antibacterial and antioxidant ability of β‐cyclodextrin complexes of vaporized Illicium verum essential oil

Abstract Compared with traditional liquid–liquid embedding method and solid–liquid embedding method of inclusion complexes of β‐cyclodextrin (β‐CD) inclusion of essential oil to form stable properties, the gas–liquid embedding method was applied to encapsulate vaporized illicium verum essential oil (IvEO), with β‐CD as wall materials so that core and wall materials molecules are in active state during complexing process. At optimal conditions with a mass ratio of 1:10, temperature of 80°C, time of 1 h, the β‐CD‐IvEO inclusion complexes (β‐CD‐IvEO‐ICs) had an encapsulation efficiency (EE) of 84.55 ± 2.31%. Fourier transform infrared spectroscopy (FTIR) revealed the encapsulation of IvEO into inclusion complexes, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) demonstrated the enhanced thermal stability of IvEO after embedding. Furthermore, the reducing power and 2‐phenyl‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (PTIO)‐scavenging capacity displayed certain capacity of antioxidation in a short time but stronger antioxidative activities as reaction time was extended. The diameter of growth zone (DGZ) indicated stronger antibacterial activity of β‐CD‐IvEO‐ICs against Escherichia coli, Bacillus subtilis, Staphylococcus epidermidis, and Staphylococcus aureus. Moreover, the β‐CD‐IvEO‐ICs could induce the bacteria producing more reactive oxygen species (ROS) than IvEO, resulting in bacterial death.

volatile oil. However, IvEO, which has poor water solubility, high volatility, unstable physicochemical properties, and unpleasant smell, shows a relatively low availability in the application of food industry.
Considering the limitation of IvEO, there are increasing researches on preparing inclusion complexes to improve its physical and chemical properties. Zhang et al. reported that selective encapsulation of star anise essential oil (SAEO) by hydroxypropylβcyclodextrin could reduce the irritating smell of SAEO, and improve the inhibition effect of SAEO on Rhizopus stolonifer, Saccharomyces cerevisiae, and E. coli and its antibacterial stability .
IvEO could also be encapsulated by chitosan to enhance antifungal and antiaflatoxigenic potency (Dwivedy et al., 2018). The transanethole/β-cyclodextrin inclusion complexes could be evenly dispersed in the gelatin-based edible films with appropriate addition, which improved the tensile strength and surface hydrophobicity and reduced the moisture content of the edible films (Ye et al., 2017). Therefore, suitable wall materials and effective methods could improve various properties of IvEO. β-CD, as a wall material, has the advantages of masking undesired smell or taste, preventing essential oil from being oxidized (Hill et al., 2013;Kavetsou et al., 2021), and increasing the water solubility (Celebioglu et al., 2018) as well as being nontoxic and of low cost (Beirão da Costa et al., 2012).
The method of increasing diffusion forces between gaseous cinnamon essential oil and β-CD solution so that core and wall materials molecules are in active state during complexing process has been studied (Zhang, 2017). Moreover, antibacterial and antioxidant properties of essential oils (EOs) before and after encapsulation have been extensively studied (Raksa et al., 2017). Nonetheless, the characterization of inclusion complexes that were prepared by the gas-liquid embedding method, antioxidant activity during sustained release process of inclusion complexes and its antibacterial mechanism have been rarely studied. Thus, this study aimed to prepare and characterize the β-CD-Illicium verum EO inclusion complexes (β-CD-IvEO-ICs) and compare the different antioxidant activities between IvEO and β-CD-IvEO-ICs through the ferric reducing antioxidant power (FRAP) assay and 2-phenyl-4,4,5,5-tetramethylimidaz oline-1-oxyl-3-oxide (PTIO)-scavenging assay. Additionally, the longterm antibacterial activity of IvEO and β-CD-IvEO-ICs against E. coli, B. subtilis, S. epidermidis, and S. aureus was evaluated and compared.

Illicium verum essential oil (IvEO) was obtained from Xiangsi
Xinqing Health Technology Co., Ltd. Nutrient agar was supplied by Guangdong Huangkai Microbial Sci. &Tech. Co., Ltd. Total antioxidant capacity assay kit was supplied by Suzhou Grace Biotechnology Co., Ltd. Reactive oxygen species (ROS) assay kit was supplied by Applygen Technologies Inc., Beijing, China. PTIO• (CAS 18390-00-6) was supplied by Biohonor Technology Co., Ltd. All other agents used for experiment were of analytical grade. Deionized water was used to perform all experiments.

| Preparation of β -CD-IvEO-ICs
The β-CD-IvEO-ICs were prepared as previously described with slight modifications (Zhang, 2017). The β-CD solution was prepared using hot distilled water with water:β-CD ratio of 9:1. The extraction device is shown in Figure 1. High temperature steam, evaporated from distillation flask, was led to a 3-neck boiling flask and the IvEO heated. After IvEO (core material) completely evaporated, it led into a β-CD solution, and kept sealed -heating at a certain temperature with stirring for a certain time. The mixture was then kept stirring at 300 r min −1 and 25°C for 5 h and the complexes were dried at 40°C for 4 h after vacuum filtration. The β-CD-water-ICs were prepared in the same way with water as the core material.

| The effects of the preparation conditions on the EE
The inclusion complexes' preparation conditions were controlled to evaluate the influences of the ratio of oil to wall materials, temperature, and time on the encapsulation efficiency (EE) through a singlefactor experiment (Cui et al., 2021).

Practical Application
β-CD-Illicium verum essential oil inclusion complexes (β-CD-IvEO-ICs) can prevent IvEO from being oxidized and increase its antibacterial activity so that β-CD-IvEO-ICs could be employed as a potential natural preservative in food industry.

| Time
The β-CD-IvEO-ICs preparation conditions were controlled to evaluate the time condition (0.5, 1, 1.5, 2, 2.5, and 3 h) on the EE with the oil-wall ratio of 1:10 at 300 r min −1 for 1 h.

| Orthogonal design
The ratio of oil to wall materials, time, and temperature conditions were selected as three influential factors (marked as A, B, and C).
Influential factors were investigated at three levels. EE was used as the evaluation index to confirm the optimum process parameter.

| Fourier transform infrared spectroscopy
The Fourier transform infrared spectroscopic (FT-IR) assay was conducted as described earlier with some modifications (Yang et al., 2021).
The β-CD, IvEO, β-CD, and IvEO physical mixture and β-CD-IvEO-ICs were mixed separately with potassium bromide (KBr) and abraded and pressed for slice formation. The spectra were obtained in the infrared (IR) region of 4000-400 cm −1 through Fourier transform infrared spectroscopy (FTIR) (Nicolet 6700 FTIR, Thermo Fisher Scientific), with a scanning number of 32 and resolution of 1 cm −1 .

| EE determination
The IvEO content was determined using a spectrophotometer according to the method described previously with some modifications (Ghazy et al., 2021;Zhang, 2017). Different concentrations of IvEO standard solutions (1, 2, 3, 4, 5, and 6 μl L −1 ) were prepared using absolute ethanol, and absorbance of the solutions was measured at 259 nm with absolute ethanol as the blank control. The IvEO concentration and absorbance were taken as the abscissa and ordinate, respectively. The obtained standard calibration curve equation was: Y = 0.1259x − 0.0014, R 2 = 0.9998.
The inclusion complexes particles (0.02 g) were added into a 10 ml volumetric flask and constant volume with ethanol. Surface oil in the inclusion complexes was washed by 10 ml ethanol. The total oil of β-CD-IvEO-ICs was dissolved using 1 ml hot water and 200 μl absolute ethanol with stirring at 300 r min −1 for 2 min. Absorbance values of all the solutions were determined using an ultraviolet-visible (UV-Vis) spectrophotometer (TU-1950, Beijing Purkinje General Instrument Co., Ltd.) at 259 nm. The amount of IvEO was estimated using the standard calibration curve. The EE was calculated as following Equation (1): where W 1 is the mass (g) of oil on the β-CD-IvEO-ICs surface and W 2 is the total mass (g) of oil into β-CD-IvEO-ICs.
After 10 μl IvEO/β-CD equivalents of the samples, 90 μl 80% ethanol and 100 μl ultrapure water were added to 850 μl FRAP reagent, the mixture was incubated at 25°C for different times (10 min and 1 h). Absorbance values of all the solutions were determined using a UV-Vis spectrophotometer (TU-1950, Beijing Purkinje General InstrumentCo., Ltd.) at 590 nm. The reducing power of samples was estimated using the standard calibration curve, and the results are presented as Trolox equivalent antioxidant capacity (μmol Trolox 100 g −1 ) (Cuong et al., 2020). 2.6.2 | 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO·)-scavenging activity measurements of IvEO and IvEO-NPs The methodology for determining the radical-scavenging ability referred to Li et al. with slight modifications (Li et al., 2021). In brief, 10 μl IvEO/β-CD equivalents of IvEO, β-CD-IvEO-ICs, β-CD, and physical mixture were prepared with 750 μl deionized water and 1.2 mg ml −1 PTIO solution was prepared with deionized water. One thousand 1000 μl of the oil solutions was mixed with 90 μl PTIO soution with essential oil free as blank control. Absorbance of samples and controls was measured at 557 nm after mixing and incubation in dark at 25°C for different times (2 h, 1 day, 2 days, 3 days, 4 days, 5 days, and 6 days) with TU-1950 UV-Vis spectrophotometer. The percent inhibition of the PTIO radical by the samples was calculated according to Equation (2): where A blank is absorbance without sample, A reaction mixture is absorbance with sample, and A sample is absorbance with sample.

| Microorganisms
Strains were streaked on Nutrient agar plates and placed in a 37°C incubator for 24 h. A single colony was picked from the plate and bacterial suspension with absorbance of 3.0 at 600 nm was prepared with TU-1950 UV-Vis spectrophotometer.

| Growth zone measurement
The zones of growth for bacteria under IvEOs treatment were determined using the Oxford cup plate assay. The methodology was used with some modification in the method described earlier (Yang et al., 2021), which is adding bacterial suspension into Oxford cups to make it grow and divide on the IvEOs plates. A study showed that agar is generally formed as a polyelectrolyte gel and the resulting 3D polymeric network has porous structure, which allows the liquid to diffuse and predicted that essential oil in a Petri dish might be relevant to its diffusion coefficient into agar medium (Mutlu-Ingok et al., 2020).
Because of the low diffusion rate of EO, adding EO into Oxford cups may result in a phenomenon that bacteria have already grown on the surface before EO diffuses into the surface of agar plates, which causes a poor antibacterial effect. Therefore, Oxford cups were always taken out after the medium solidified, and then the antibacterial substance was loaded into the holes so that it can come into contact with the bacteria directly according to a study (Yang et al., 2018). On the contrary, bacteria could grow normally in the agar by adding bacterial suspension into cups and antibacterial substances have already diffused before exposing to bacteria, which means it offers no length of diffusion time issues for antibacterial substances. Subsequently, two treatment groups were designed to evaluate the antibacterial activity of IvEO and β-CD-IvEO-ICs. The IvEO concentration of the β-CD-IvEO-ICs groups was adjusted to that of the IvEO groups.
IvEO and β-CD-IvEO-ICs were added into Nutrient agar (cool to 40°C) respectively. First, 10 ml of molten Nutrient agar was added into sterilized plates until solidification. Then, another 10 ml of molten Nutrient agar was poured on the plates. Afterwards, two Oxford cups were placed on each Nutrient agar plate. After cooling and solidification of Nutrient agar, each bacterial suspension was added into one of the cups and sterile water was added into another cup as a negative control. The culture dishes were incubated at 37°C for 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 days to observe differences in strain growth in IvEO and β-CD-IvEO-ICs plates. The diameter of each inhibition zone was measured using cross method. The antibacterial activity of IvEO and β-CD-IvEO-ICs was estimated by measuring the diameter of growth zone (DGZ).

| Detection of ROS
In brief, 20 ml of molten Nutrient agar (cool to 40°C) was mixed with certain amount IvEO and β-CD-IvEO-ICs and poured on the plates with sterile water as blank control. Then, the bacterial suspension (50 μl) with absorbance of 3.0 at 600 nm was inoculated onto Nutrient agar plates in a 37°C incubator for 24 h. Afterwards, the treated bacteria were collected by washing the plate using 5 ml phosphate-buffered saline (PBS). After the suspension adjusted to the absorbance of 0.200 ± 0.005 at 600 nm then triple diluted, 3 μl of 10 mmol L −1 dichlorodihydrofluorescein diacetate (DCFH-DA) was added into the mixture in dark at 37°C for 30 min and the mixture was shaken every 5 min.
The dichlorofluorescein (DCF) fluorescence of the mixture mentioned above was measured using a fluorescence spectroscope (2)

| Statistical analysis
All experiments were performed at least in triplicate and data are expressed in means ± standard deviations (n > 3). Statistical analyses were performed using Origin 2018.

| Effects of preparation conditions on the EE
3.1.1 | Ratio of oil to wall materials As shown in Figure 2, the yield of β-CD-IvEO-ICs increased significantly, reaching the highest value at the oil:wall material mass ratio of 1:10 (w/w), followed by a decrease. At high concentration of oil material, the free oil adhered to the surface of β-CD-IvEO-ICs, which causes their aggregation to lower the EE. Increasing the ratio of wall material, which means more IvEOs can be fully immersed in the hydrophobic cavity of β-CD, could gradually increase the EE which is consistent with previous studies. However, the saturation of β-CD by further embedding of IvEO would decrease the EE of inclusion complexes. This was probably due to a dynamic equilibrium during complexing process between β-CD and IvEO in aqueous solutions.

The equation of dissociation and inclusion was shown as follows (Equation [3]):
Namely, such an encapsulation method resulted in the encapsulation efficiency that cannot reach 100%. The result was also observed in a study, which showed that some content of a core material can be found on the outer surface or some areas that are close to the surface of an encapsulate at a higher load (Jafari et al., 2008). According to a report, the hydrophobic cavity that wall material provided during the complexing process is not completely occupied, which means the relationship of nonstoichiometric host molecule and guest molecule is available (Sun, 2012).
It can be considered that the complexing process has achieved equilibrium at an oil:wall material mass ratio of 1:10 (w/w), which means there is not enough essential oil for further inclusion with the mass of wall material increases. After that, the mass of free oil can be considered as an invariable constant. According to Equation 3, on the basis of invariable mass of free oil, decreasing the mass of whole essential oil can decrease the EE, which causes the EE of complexing process to hardly reach 100% in aqueous solutions. When the temperature value ranged from 80 to 100°C, the EE of β-CD-IvEO decreased gradually probably due to anethole, as the main ingredient of IvEO, which could gradually react with oxygen at 55 ~ 95°C and increase its oxidation reactivity which lead to the reduction of oil material (Zhang, 2018).  (Zhang, 2018). It can be stated that excessive reaction time may lower the EE.

| Analysis of the orthogonal design
As shown in Table 1, the R-value decreased in the order: Accordingly, the influence of each factor on EE in decreasing order was as follows: time value > the ratio of oil to wall materials > temperature value. Obviously, the time value had great influences on the EE of β-CD-IvEO-ICs. When the mass ratio of oil to wall materials, Effect of the ratio of oil to wall materials on the encapsulation efficiency (EE).

| SEM
It can be noted in Figure 5(a,b,e, and f) that β-CD-water-ICs were regular, translucent block, and cracked. β-CD-IvEO-ICs were amorphous fine particle and crack-free. As shown in Figure 5(c,d,g, and h), β-CD-water-ICs were compact and big crystal. However, β-CD-IvEO-ICs was small aggregate with lots of particulate by loose conglomeration, which was obviously different from β-CD-water-ICs.
According to a report, β-CD-water-ICs consisted solely of β-CD molecule, in which intramolecular or intermolecular hydrogen bonds between the C2 and C3 hydroxyl form hydrogen-bond-type crystal lattice (Prabhu et al., 2018). After β-CD forms inclusion with IvEO molecule, the IvEO molecule can interfere with the formation of hydrogen bonds, and change the molecular arrangement or accumulation to affect changes in crystal morphology. The results are in line with those of a study (Hua.Chai et al., 2011).

| FTIR features measurement
The FTIR was applied to evaluate the EE based on the structure and complexes, β-CD and physical mixture had great difference between 1200 and 1600 cm −1 which display the variation of C-H absorption (Wang et al., 2011b). This was probably due to the fact that IvEO molecules combined with the hydrophobic bond of β-CD molecules (Haiyee et al., 2009) under the impetus of van der Waals force and hydrophobic interaction (Yang et al., 2009), which promoted the formation of β-CD-IvEO-ICs.

F I G U R E 3
Effect of the temperature condition on the encapsulation efficiency (EE). for IvEO were related to its endothermic decomposition (Figure 7a).
The thermogram of β-CD showed a wide endothermic peak at about 90 ~ 100°C (Figure 7b), which could be related to the dehydration of water molecules that bind to β-CD molecules (Marini et al., 1996).
Besides, there was an intensive peak at 313.85°C that derived from
Interestingly, the reducing power of β-CD-IvEO-ICs was higher than that of IvEO after 1 h reaction time, which could be related to the complexing process.
As shown in Figure 9, β-CD-IvEO-ICs, IvEO, physical mixture, and β-CD had certain radical-scavenging capacity. The radicalscavenging rates of IvEO, physical mixture, and β-CD were basically saturated within 2 h. But the PTIO-scavenging rate of β-CD-IvEO- In the radical-scavenging rate curves (Figure 9), the physical mixture showed higher PTIO· scavenging capacity than IvEO and β-CD also had the ability of scavenging radical. But β-CD had weak reducing power. This phenomenon could be attributed to the difference between PTIO· and Fe3 + -TPTZ (2,4,6-tripyridyl-s-triazine) as core materials. A study showed that β-CD can selectively bind aromatic compounds with suitable shape and size to form supramolecular system (Jiang, 2011). PTIO, as an aromatic derivation, was probably partially complexed in β-CD during scavenging process, which causes a drop in the concentration of PTIO· solution. It is difficult for the radical that was complexed to react with the outside world so that it could be considered a way to scavenge radical, which meant that β-CD had radical-scavenging capacity in appearance.

TA B L E 2
The reducing power of the β-cyclodextrin-illicium verum essential oil inclusion complexes (β-CD-IvEO-ICs), IvEO, anethole, β-CD by using the ferric reducing antioxidant power (FRAP) method in 10 min that strong antibacterial activity of IvEO was conserved for a long time due to the less loss embedded IvEO whose volatile components were protected (Marino et al., 2001).
In addition, bacteria might build up a tolerance to essential oil, which means that when IvEO possessed an inhibitory effect to bacteria but not enough to kill them, they might develop a tolerance to IvEO and transmit the information to the offspring. For S. epidermidis and E. coli, the 10-day culture DGZ of IvEO was shown to be comparable to the DGZ of sterile water. The phenomenon, which presented inhibition first then growth normally, was probably because bacteria had tolerance to IvEO for generations. However, the 10-day culture DGZ of β-CD-IvEO-ICs was smaller than sterile water for four targeted bacteria. This was probably because bacteria would not develop a strong tolerance when they were exposed to IvEO with low concentration due to the controlled release of complexed IvEO. Besides, β-CD-IvEO-ICs could inhibit the growth of bacteria for generations as the concentration increased which means inhibition of β-CD-IvEO-ICs on bacteria is a long-term and effective process.
3.5.2 | Excessive ROS production in IvEOtreated and β-CD-IvEO-ICs-treated bacteria The methodology used to cultivate bacteria was described in section 2.7.3, which presents that the higher addition amount of IvEOs, the lower was the bacterial count. Futhermore, when IvEOs content added was 48 μl, plates of β-CD-IvEO-ICs demonstrated tiny amounts of bacteria and only the growth of S. aureus could be seen in the plates of IvEO.
As illustrated in Figure 10 and Table 5  wall in quantities with increase in the addition of IvEOs, can program bacteria to produce excessive ROS. The excessive oxidative ROS may damage the bacteria and cause bacterial lysis to death (Lijun et al., 2021). As mentioned above, the mechanism of IvEOs was similar to antibiotics which stimulate the production of highly deleterious hydroxyl radicals in bacteria through drug-target interaction (Kohanski et al., 2007). The differences of bacterial ROS production level between IvEO and β-CD-IvEO inclusion complexes were measured by 2′,7′-dichlorofluorescein diacetate (H 2 DCF-DA). As shown in Table 5, the DCF fluorescence of β-CD-IvEO inclusion complexes was significantly higher than that of IvEO, which is probably due to its increased water solubility that led to increased contact area between the bacteria and essential oil, thus improving antimicrobial activity and stimulating the bacteria to produce more excessive ROS . A study also showed that lethal action derives from stimulation of a self-amplifying accumulation of ROS that overwhelms the repair of primary damage (Hong et al., 2019). The result is in line with section 3.5.1, which means that complexed IvEO can induce more excessive ROS production in bacteria than IvEO equivalents due to the higher water solubility and stability. In addition,

| CON CLUS ION
Vaporized IvEO was encapsulated through gas-liquid embedding method with β-CD as wall materials and the IvEOβ-CD-ICs exhibited the high EE of 84.55 ± 2.31%. Results of FTIR, DSC, and TG measurements demonstrated that the vaporized IvEO had encapsulated into IvEOβ-CD-ICs and that the stability of IvEOβ-CD-ICs was improved. In addition, the antioxidative activities of IvEOβ-CD-ICs were slightly decreased compared with that of IvEO during the early period of reaction, whereas IvEOβ-CD-ICs demonstrated long-lasting antioxidative activities and antibacterial activities against E. coli, B. subtilis, S. epidermidis, and S. aureus during the whole period. Therefore, we suggest that IvEOβ-CD-ICs could be used as a natural preservative. However, further studies are required to improve the quality of food with IvEOβ-CD-ICs during storage and investigate the preservation effects on tested food.

ACK N OWLED G EM ENTS
The authors acknowledge the financial support of Forestry Scientific  19ZK0364).

CO N FLI C T O F I NTE R E S T
There are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.